Cell-containing preparations

ABSTRACT

The present invention provides a cell-containing preparation comprising cells containing a DNA having a base sequence represented by SEQ ID NO:1 or 2, or a DNA hybridizable with a DNA having a base sequence represented by SEQ ID NO:1 or 2 under stringent conditions, and a fibrous protein. The cell-containing preparation of the present invention makes it possible to more efficiently supply NK4 having an inhibitory action against the growth or metastasis of cancers to cancer cells.

TECHNICAL FIELD

The present invention relates to a cell-containing preparation forproviding NK4 to prevent and treat the growth, invasion and metastasisof cancers with use of an NK4-encoding DNA. In particular, the presentinvention provides a cell-containing preparation for preventing andtreating the growth, invasion or metastasis of cancers by supplying NK4secreted from cells transformed with an NK4-encoding DNA to cancer cellsor cancer tissues, a method for inhibiting the growth, invasion ormetastasis of cancers using the said preparation, and a method forproducing the said preparation.

BACKGROUND ART

Complete cure for cancers which readily undergo metastasis, such aspancreatic cancer, is difficult, and excision of organs at the affectedpart by cancers has been considered to be only an effective therapeuticmethod. However, since cancer cells which readily undergo metastasisrapidly invade into the surrounding tissues around the cancer cells, themetastatic cancer recurs with a quite high frequency when prognosis isinsufficient after excision by surgery (see, for example, Takeda, “Therole of adjuvant therapy for pancreatic cancer”, Hepatogastroenterology,USA, 2001, Vol. 48, No. 40, p 953-956; Cienfuegos J A, Analysis ofintraoperative radiotherapy for pancreatic carcinoma, Eur J Surg Oncol.,United Kingdom, 2000, Vol. 26-A, S13-15). Most of the causes of death bythe cancer are not directly related to primary cancers, but are due tothe recurred metastatic cancers.

Radiotherapies and chemotherapies such as administration of anticanceragents are usually used for the treatment of the cancer, and atherapeutic method by excision surgery of the tissues with an aid of thechemotherapy has been attempted in view of preventing the recurrence ofcancers which readily undergo metastasis. Although inhibitory effects oncancer have been recognized in the radiotherapy and chemotherapy such asadministration of anticancer agents, these therapeutic methods cannot bealways considered to be safe, since they involve risks of variousadverse effects such as myelosuppression, loss of hair, injuries of theheart and nerve, and injuries of the digestive tract and liver.

Accordingly, development of not only therapeutic methods of inhibitingproliferation of the primary cancer itself but also a method forpreventing the onset of metastatic cancers have been urgently desired inthe treatment of cancers. Furthermore, any therapeutic methods thatsatisfy both requirements without any adverse effects while being safefor the living body have not yet been established.

Hepatocyte growth factor (HGF) is a hetero-dimer protein composed of anα-chain and β-chain. It has been reported from many studies that HGFexpresses a mitogenic activity, motogenic activity and morphogenicactivity by binding to a c-Met/HGF receptor, and results in exhibitingactions of inducing invasion or metastasis of tumor cells, andangiogenesis(see, for example, Kunio Matsumoto et. al, “HGF in lungmorphogenesis and tumor infiltration: role as a mediator inepithelium-mesenchyme and tumor-stroma interactions”, Cancer Chemother.Pharmacol., 1996, Vol. 38, Suppl, S42-7; Jiang. W. G et al, “HGF/scatterfactor, its molecular, cellular and clinical implication in cancer”,1999, Crit. Rev. Oncol. Hematol, Vol. 29, p 209-248).

NK4 is a peptide comprising an N-terminal hair-pin domain of the α-chainof HGF and four kringle domains, and acts as an antagonist of HGF byinhibiting the HGF from binding to the c-Met/HGF receptor (see, forexample, Date K. et al., “Inhibition of tumor growth and invasion byfour-kringle antagonist (HGF/NK4) for HGF”, 1998, Oncogene, Vol. 17, No.23, p 3045-54; Parr C., et al., The HGF/SF-induced phosphorylation ofpaxillin, matrix adhesion, and invasion of prostate cancer cells wereinhibited by NK4, an HGF/FS variant, 2001, Biochem. Biophys. ResCommun., Vol 285, No. 5, p 1330-7). NK4 is known to inhibit growth,invasion or metastasis of tumors by its antagonist activity against HGF,and to inhibit the angiogenesis induced by HGF as well as by VEGF andbFGF through a mechanism different from that for the antagonist activityagainst HGF (see, for example, Kuba K., et al., “Kringle 1-4 of HGFinhibits proliferation and migration of human microvascular endothelialcells”, 2000, Biochem. Biophys. Res. Commun., Vol. 279, No. 3, p846-52).

From the above facts, the growth and metastasis of cancers are inhibitedby supplying NK4 to the cancer tissue. Furthermore, NK4 can be safelyadministered to the living body since it is a protein derived from theliving body.

The methods for supplying NK4 to the cancer tissue include a method foradministering NK4 itself, or a method using a gene therapy technologycomprising directly introducing a NK4-encoding gene into cancer cells toproduce NK4 therein. The method for directly introducing NK4 itself intothe cancer cells is preferable for the reason that it can exhibit animmediate effect with an expectation of high therapeutic efficiency,however, the procedure of this method is complicated because it requiresprocesses for cultivating a large amount of NK4-producing cells and forextracting and purifying NK4 from the cells.

On the other hand, the method for introducing the NK4-encoding gene intothe cancer cells is advantageous in that NK4is not required to beproduced and purified in advance. Means generally used for introducingthe gene into the cancer cells is to integrate the NK4-encoding geneinto a vector such as an adenovirus vector, and to transfect the cancercells with the vector.

The methods for introducing a gene encoding a protein effective fortreating the cancer or the protein itself effective for treating thecancer into the cancer cells have been disclosed to date. These methodscomprise transfecting the cancer cells with a vector by contact with thecancer tissue by covering the cancer tissue with light-curable gelatinin which the vector having the gene or the protein itself is contained,or allowing the protein to permeate into the cancer tissue (see, forexample, Jain R K., et al., “Barriers to drug delivery in solid tumors”,1994, Sci Am., Vol. 271, No. 5, p 498-504) . While the protein waspossible to be efficiently permeated into the cancer cell by the methodabove, it was difficult to effectively introduce the gene into thecancer cells since permeability of the vector as a large molecule intothe cancer cells was low. While it may be more advantageous to supplythe protein itself to the cancer cells from the above facts, this methodcannot be always considered to be an excellent technology in view of itspractical use because the protein should be independently produced andpurified as described above.

Technologies that are able to practically and efficiently supply NK4 tothe cancer cell such as the cell-containing preparation of the presentinvention have not been known in the past. In other words, thecell-containing preparation of the present invention is a technologyinvolving both advantages of administering NK4 itself and of producingNK4 in cancer cells by introducing an NK4-encoding gene into the cancercells directly. NK4 may be supplied to the cancer cells in the form of aprotein having high permeability to the cancer cells without anynecessity of independently producing and purifying NK4. Moreover, thepreparation may be formed into a shape suitable for administrationsites, for example the entire area of the cancer tissue as a target canbe covered by forming the preparation into a sheet. Metastasis andrecurrence of the cancer that has been left behind by surgery and iscaused by the primary cancer may be also prevented by covering theremaining portions of the cancer after excision by surgery.

The cell-containing preparation of the present invention has no risks ofonset of adverse effects as occur by using the chemotherapy as describedabove, and may be a safe anticancer agent or a cancer metastasisinhibitor for patients when the preparation is produced using patient'sown cell samples from the patient.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a cell-containingpreparation having an inhibitory effect for growth of cancers ormetastasis and recurrence of cancers and having a safety to livingbodies, in particular a cell-containing preparation capable of moreefficiently supplying NK4 having an inhibitory action for growth ormetastasis of cancers to cancer cells. Another object of the inventionis to provide a method for inhibiting the growth or metastasis ofcancers or for inhibiting the angiogenesis, using the cell-containingpreparation. A still another object of the invention is to provide amethod for producing the cell-containing preparation.

Cells containing a DNA having a base sequence represented by SEQ ID NO:1 or 2, or a DNA hybridizable with a DNA having a base sequencerepresented by SEQ ID NO:1 or 2 under stringent conditions are shapedwith a fibrous protein, for example, collagen to form a complex, andthis complex was transplanted around the cancer tissue. The inventors ofthe present invention have found, through intensive studies forattaining the above-mentioned objects, that NK4 produced in the cells inthe form of complex can be more effectively supplied to the cancertissue in proper quantities, and have completed the invention throughadditional studies based on these findings.

The present invention provides:

(1) A cell-containing preparation comprising a cell which has a DNAhaving a base sequence represented by SEQ ID NO: 1 or 2 or a DNAhybridizable with a DNA having a base sequence represented by SEQ ID NO:1 or 2 under stringent conditions and a fibrous protein;

(2) the cell-containing preparation according to the above (1), whereinthe cell is an epithelial cell of the oral mucosa, a skin cell or afibroblast;

(3) the cell-containing preparation according to the above (1) or (2),wherein the fibrous protein is collagen;

(4) the cell-containing preparation according to any one of the above(1) to (3), wherein the cells are deposited on the surface of thefibrous protein;

(5) the cell-containing preparation according to any one of the above(1) to (4), wherein the cell is a transformant;

(6) the cell-containing preparation according to the above (5), whereinthe transformant is transformed with a recombinant expression vector;

(7) the cell-containing preparation according to the above (6), whereinthe recombinant expression vector is adeno-associated virus (AAV),retrovirus, poxvirus, herpes virus, herpes simplex virus, lentivirus(HIV), Sendai virus, Epstein-Barrvirus (EBV), vaccinia virus,poliovirus, sindbis virus, SV40 or plasmid;

(8) the cell-containing preparation according to any one of the above(1) to (7) capable of forming a peptide encoded by a DNA having a basesequence represented by SEQ ID NO:1 or 2, or by a DNA hybridizable witha DNA having a base sequence represented by SEQ ID NO:1 or 2 understringent conditions;

(9) the cell-containing preparation according to any one of the above(1) to (8) further containing a mesh sheet comprising a biodegradableresin;

(10) the cell-containing preparation according to the above (9), whereinthe biodegradable resin is polyglycolic acid;

(11) the cell-containing preparation according to any one of the above(1) to (10), which is an anticancer agent or a cancer metastasisinhibitor;

(12) the cell-containing preparation according to the above (11), whichis an anticancer agent or a metastasis inhibitor for ovarian cancer,pancreatic cancer, stomach cancer, gall bladder cancer, kidney cancer,prostate cancer, breast cancer, esophageal cancer, liver cancer, oralcavity cancer, colon cancer, large intestine cancer, sarcoma, glioma ormelanoma;

(13) the cell-containing preparation according to any one of the above(1) to (10), which is an angiogenesis inhibitor;

(14) a method for inhibiting growth, invasion and metastasis of cancersor for inhibiting angiogenesis, which comprises administering thecell-containing preparation according to any one of the above (1) to(13) to a mammal;

(15) a method for producing a cell-containing preparation whichcomprises culturing a cell on the surface of a fibrous protein andtransforming the cultured cells with a recombinant expression vectorcomprising a DNA having a base sequence represented by SEQ ID NO: 1 or2, or with a recombinant expression vector comprising a DNA hybridizablewith a DNA having a base sequence represented by SEQ ID NO:1 or 2 understringent conditions;

(16) a method for producing a cell-containing preparation , whichcomprises preparing a fibrous protein sheet by coating a fibrous proteinonto a mesh sheet comprising a biodegradable resin; culturing a cell onthe surface of the fibrous protein sheet obtained; and transforming thecultured cells with a recombinant expression vector comprising a DNAhaving a base sequence represented by SEQ ID NO:1 or 2, or with arecombinant expression vector comprising a DNA hybridizable with a DNAhaving a base sequence represented by SEQ ID NO:1 or 2 under stringentconditions; and

(17) a method for producing a cell-containing preparation, whichcomprises transforming the cells with a recombinant expression vectorcomprising a DNA having a base sequence represented by SEQ ID NO:1 or 2,or with a recombinant expression vector comprising a DNA hybridizablewith a DNA having a base sequence represented by SEQ ID NO:1 or 2 understringent conditions, and mixing the resulting transformant cells with afibrous protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing an example of the cell-containingpreparation of the present invention and a transplantation methodthereof.

FIG. 2 is a schematic drawing showing recombinant adenovirus vectorAd-NK4.

FIG. 3 shows an example of an SEM analysis photograph (cross-section) ofthe cell-containing preparation of the present invention.

FIG. 4 is a graph obtained by monitoring time-dependent NK4 secretion ofOMEC into which Ad-NK4 is introduced.

FIG. 5 shows an effect of introduction of Ad-NK4 on the growth of OMEC.

FIG. 6 is a graph showing an effect of NK4 secreted from into whichAd-NK4 is introduced, on inhibition of invasion of pancreatic cancercells.

FIG. 7 is a graph showing concentrations of NK4 in the tumor and in theserum of mouse tumor model transplanted with an example of thecell-containing preparation.

FIG. 8 is a graph showing an effect for inhibiting the tumor volume fromincreasing in mouse tumor model transplanted with an example of thecell-containing preparation.

FIG. 9 is a graph showing an effect for inhibiting the number ofangiogenesis from increasing in the mouse tumor model transplanted withan example of the cell-containing preparation.

In the drawings, the reference numeral 1 denotes collagen, the referencenumeral 2 denotes VICRYL™ mesh sheet, the reference numeral 3 denotesOMEC, the reference numeral 4 denotes OMEC into which Ad-NK4 isintroduced, the reference numeral 5 denotes a gene product (NK4), thereference numeral 6 denotes a cancer tissue, and reference numeral 7denotes a tissue.

BEST MODE FOR CARRYING OUT THE INVENTION

The cell-containing preparation of the present invention comprises acell having (a) DNA having a base sequence represented by SEQ ID NO: 1or 2 or (b) a DNA hybridizable with a DNA having a base sequencerepresented by SEQ ID NO: 1 or 2 under stringent conditions, and afibrous protein.

The base sequence represented by SEQ ID NO: 1 or 2 is an example of theNK4-encoding DNA. The bases from base No. 391 to base No. 405 in thebase sequence represented by SEQ ID NO: 1 are deleted in the basesequence represented by SEQ ID NO: 2, and the protein produced by thisDNA also has an antagonist activity against HGF and an inhibitoryactivity against angiogenesis.

The DNA hybridizable with a DNA having the base sequence represented bySEQ ID NO: 1 or 2 under stringent conditions in the present inventionmeans, for example, a DNA obtained by using a colony hybridizationmethod, plaque hybridization method or Southern hybridization method,using the above DNA as a probe. Specifically, the DNA may be identifiedby hybridization at about 65° C. in the presence of about 0.7 to 1.0 Msodium chloride using a filter on which DNA derived from a colony orplaque is immobilized, followed by washing the filter at about 65° C.using a 0.1× to 2×SSC solution (the composition of 1×SSC solutioncomprises 150 mM sodium chloride and 15 mM sodium citrate).

Specific example of the DNA hybridizable with the DNA having the basesequence represented by SEQ ID NO: 1 or 2 is a DNA containing a basesequence having not less than 70%, preferably not less than 80%, morepreferably not less than 90%, and most preferably not less than 95% ofhomology to the base sequence represented by SEQ ID NO:1 or 2. Morespecifically, the DNA sequence is partially modified by deleting,substituting or adding one or more bases in the base sequencerepresented by SEQ ID NO:1 or 2 by various artificial processing, forexample by site-specific mutagenesis, random mutation by treating with amutagenesis agent or digestion with a restriction enzyme, so long assuch modified DNA is able to produce a peptide having an antagonistactivity against HGF and an inhibitory action against angiogenesis.

Hybridization may be performed by the known method, for example, by themethod described in Molecular Cloning, A laboratory Manual, ThirdEdition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001:abbreviated as Molecular Cloning, Third Edition). When commerciallyavailable libraries are used, the method may follow the technicalinstruction attached.

Specific examples of the DNA having the base sequence in which one ormore bases are deleted, substituted or added in the base sequencerepresented by SEQ ID NO: 1 or 2 is (a) a base sequence in which one ortwo or more (preferably about 1 to 30, more preferably about 1 to 10,and more preferably several (about 1 to 5)) bases are deleted in thebase sequence represented by SEQ ID NO:1 or 2, (b) a base sequence inwhich one or two or more (preferably about 1 to 30, more preferablyabout 1 to 10, and more preferably several (1 to 5) bases are added inthe base sequence represented by SEQ ID NO:1 or 2, (c) a base sequencein which one or two or more (preferably about 1 to 30, more preferablyabout 1 to 10, and more preferably about 1 to 5) bases are substitutedwith other bases in the base sequence represented by SEQ IDNO:1 or 2, or(d) a base sequence having a base sequence in combination thereof.

Substitution or other operations may be applied by the known method suchas ODA-LA PCR method, gapped duplex method or Kunkel method, or a methodsimilar to these methods using PCR and known kits, for exampleMutan™-superExpress Km (Takara Shuzo Co.) or Mutan™-K (Takara ShuzoCo.).

For cloning a DNA fragment having a complete base sequence of (a) theDNA having the base sequence represented by SEQ ID NO: 1 or 2 or (b) aDNA hybridizable with the DNA having the base sequence represented bySEQ ID NO:1 or 2 under stringent conditions, the fragment is amplifiedby a PCR method using a synthetic DNA primer having a partial basesequence of (a) or (b), for example, a known HGF primer (for example aDNA fragment having a base sequence represented by SEQ ID NO:5 or 6); orthe DNA may be selected by hybridization from DNAs in which appropriatevectors are integrated using a DNA fragment or a synthetic DNA encodinga partial or whole sequence of labeled HGF protein.

The DNA may be cloned by chemical synthesis using known methods fromknown base sequence information of HGF. An example of the chemicalsynthesis method is to use a DNA synthesizer such as DNA synthesizermodel 392 (manufactured by Perkin-Elmer Co.) by the phosphoramiditemethod.

The DNA used in the present invention may be modified for enhancing itsstability in the cells or for reducing its toxicity, if any. Manymodification methods are known in the art, and may be carried outaccording to the method disclosed in Kawakami et al., Pharm Tech Japan,Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; S. T. Crookeetal. ed.,Antisense Research and Applications, CRC Press, 1993.

The 3′-end or 5′-end of the DNA used in the present invention may bechemically modified with a protective group known in the art, such aspolyethylene glycol and tetraethylene glycol in order to preventdecomposition with nucleases such as exonuclease and RNase.

The DNA may have ATG as a translation initiation codon at the 5′-end andTAA, TAG or TGA as a translation stop codon at the 3′-end. Thesetranslation initiation codons and translation stop codons may be addedto the DNA using an appropriate DNA adapter. It is preferable that theDNA has a polyadenyl sequence at the 5′-end.

An NK4-encoding RNA may be used in the present invention so long as itis able to express an antagonist activity against HGF and anangiogenesis inhibitory activity by treatment with a reversetranscriptase, and the RNA may be obtained by known methods. Such RNA isto be also included in the DNA used in the present invention.

The cell used in the present invention comprises (a) a DNA having a DNAsequence represented by SEQ ID NO:1 or 2, or (b) a DNA hybridizable withthe DNA having a DNA sequence represented by SEQ ID NO:1 or 2 understringent conditions.

The method for introducing the above DNA (a) or (b) into the cells usedin the present invention is not particularly restricted, and knownmethods may be used. For example, while such method include a method ofintroducing the DNA into the cells by allowing it to be contained inrecombinant expression vectors such as plasmids and viruses, or inartificial vectors such as liposomes and microcapsules, a recombinantexpression vector is preferably used in the present invention. Anymethods known per se may be used for introducing the recombinantexpression vector into a host. Examples of such methods include acompetent cell method [J. Mol. Biol., 53, 154 (1970)], DEAE dextranmethod [Science, 215, 166 (1982)], in vitro packaging method [Proc.Natl. Acad. Sci., USA, 72,581 (1975)],virus vector method [Cell, 37,1053 (1984)], micro-injection method [Exp. Cell. Res., 153, 347 (1984)],electroporation method [Cytotechnology, 3, 133 (1990)], calciumphosphate method [Science, 221, 551 (1983)], and lipofection method[Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)], and protoplast method[Japanese Patent Application Laid-Open No. 63-2483942, Gene, 17, 107(1982), Molecular & General Genetics, 168, 111 (1979)].

The recombinant expression vector used in the present invention ispreferably an expression vector capable of effectively producing NK4 byallowing the DNA of (a) or (b) to be expressed in the cell into whichthe expression vector is introduced. For example, a DNA fragmentcontaining the base sequence encoding NK4 is cut from cDNA, and the DNAfragment is linked to the downstream of the promoter in an appropriateexpression vector to produce the recombinant expression vector.

Examples of the expression vector available include plasmids derivedfrom Escherichia coli such as pCR4, pCR2.1, pBR322, pBR325, pUC12 andpUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5 andpC194; plasmids derived from yeast such as pSH19 and pSH15;bacteriophages such as λ phage; and animal viruses such as adenovirus,adeno-associated virus (AAV), retrovirus, poxvirus, herpes virus, herpessimplex virus, lentivirus (HIV), Sendai virus, Epstein-Barr virus (EBV),vaccinia virus, polio virus, sindbis virus and SV40; as well as pA1-11,pXT1, pRc/CMV, pRc/RSV and pcDNAI/Neo. Viruses are preferable among themas the vector used in the present invention, and retrovirus,adeno-associated virus and adenovirus are more preferable.

While adenovirus belongs to various serotypes, human adenovirus types 2or 5 are preferably used in the present invention. Use of the adenovirusvector is further preferable because adenovirus is known to have highertransfection efficiency as compared with other viruses, to be able toinfect undivided cells, and not to be integrated into genomes of cells.

The virus vector is preferably a replication-defective virus in whichvirus genes are completely or almost completely deleted. At least the E1region of the adenovirus vector is preferably non-functional. Otherregions may also be modified, and any regions of E3 region (WO95/02697), E2 region (WO 94/28938), E4 region (WO 94/28152, WO 94/12649,WO 95/02697) or late genes L1 to L5 may be particularly modified.Modified viruses such as replication-defective viruses may be producedby the method known per se. The modified viruses may also be recoveredand purified by the method known per se. The modified viruses availableare described, for example, in Japanese Patent Application NationalPublication Laid-Open Nos.11-514866, 11-506311, 9-500524, 8-501703 and8-508648, or Japanese Patent Application Laid-Open No. 8-308575.

The promoter may be any one of appropriate promoters corresponding to ahost used for expressing the gene. For example, when a mammal is used asa host, examples of the promoter include those obtained from the genomeof viruses such as Rous sarcoma virus (RSV virus), MPSV, polyoma virus,fowlpox virus, adenovirus, bovine papilloma virus, avian sarcoma virus,cytomegalovirus (SMV), hepatitis B virus, simian virus (SV40) andvaccinia virus, and metallothionein promoter and heat shock promoter.

A vector to which the following regulatory sequence is added may be usedfor the recombinant expression vector in order to allow the DNA encodingNK4 to be expressed, or in order to be advantageous for expression. Eachregulatory sequence may be either endogenous or exogenous to the vector.

While examples of the regulatory sequence include a signal sequence,promoter, pro-peptide sequence, enhancer, selection marker andterminator, it is not restricted thereto. The regulatory sequence mayhave a linker in advance so as to facilitate a linkage between the DNAencoding NK4 and the regulatory sequence, and/or a linkage between theregulatory sequences.

NK4 produced in the host cell is actively secreted out of the host cellby integrating the signal sequence into the recombinant expressionvector. Consequently, NK4 produced may be efficiently supplied to adesired site to inhibit the growth and metastasis of the cancer, andangiogenesis.

While an HGF signal sequence, an insulin signal sequence and anα-interferon signal sequence may be used as the signal sequence when thehost is mammal cells, the HGF signal sequence derived from human ispreferably used in particularly in the present invention.

The methods known per se may be used for adding the signal sequence, andexamples of such method include those described in J. Biol. Chem., 264,17619 (1989), Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), GenesDevelop., 4, 1288 (1990), Japanese Patent Application Laid-Open No.5-336963, and WO 94/23021.

An enhancer is preferably introduced into the vector when the cells ofhigher mammals, for example, human cells are used as the host.Introducing the enhancer permits transcription of the DNA inserted intothe vector to be increased. Examples of the enhancer include an SV40enhancer, a cytomegalovirus enhancer, an initial promoter/enhancer ofcytomegalovirus, a polyoma enhancer and an adenovirus enhancer.

Examples of the selection marker include dihydrofolic acid reductasegene (methotrexate (MTX) resistant), ampicillin resistant gene andneomycin resistant gene. In particular, the desired gene may be selectedin a culture medium not containing thymidine when the DHFR gene is usedas the selection marker using CHO (DHFR⁻) cells.

The cells constituting the cell-containing preparation of the presentinvention capable of producing NK4 may be produced by inserting anNK4-encoding DNA fragment into these virus vectors and transfecting thehost cells with the DNA fragment to transform the cells.

The method for preparing the virus vector, and the method for insertingthe DNA fragment into the virus vector are described in ExperimentalMedicine, Supplement Edition, Basic Technology of Gene Therapy, Yodo-ShaCo. (1996), and in Experimental Medicine, Supplement Edition,Experimental Methods of Gene Introduction & Expression Analysis,Yodo-Sha Co. (1997).

While the cells used for the cell-containing preparation of the presentinvention are not particularly restricted so long as they can serve ashost cells for the recombinant expression vector, examples of such cellsinclude animal cells, cells of the genus Escherichia, cells of the genusBacillus, Lactobacillus bifidus, lactic acid bacteria, yeast, and insectcells.

Examples of the animal cells available include epithelial cells of oralcavity mucous membrane (hereinafter abbreviated as OMEC), skin cells,fibroblasts, and somatic cells including various epithelial cells ofmammals such as human, or simian COS-7 cells, Vero, Chinese hamster CHOcells (hereinafter abbreviated as CHO cells), dhfr gene-deficientChinese hamster cells (abbreviated as CHO (dhfr⁻) cells), mouse BALB/3T3cells, mouse L cells, mouse AtT-20 cells, mouse C127 cells, mousemyeloma cells, rat GH3 cells, human HeLa cells, human FL cells, 293cells derived from the human fetal kidney (Jikken Igaku (ExperimentalMedicine), 12, 316, (1994), and various cell strains including mouseNIH3T3 cells.

The animal cells may be any one of established cells in the laboratoryand cells isolated from living tissues. The cells extracted from thetissue can be obtained by treating the tissue extracted by anappropriate means with Dispase or EDTA, followed by treating withtrypsin into single cells. The single cells obtained are then culturedto confluence in an appropriate culture medium, and a cell line isestablished by repeating passage culture twice or three times. Theestablished cells are sampled as single cells again by treating withtrypsin and collagenase, and are used as the cells of the presentinvention. The feeder layer method may be used depending on the kinds ofthe cells. For example, fibroblasts derived from mouse fetus (3T3 cells)are preferably used as feeder cells when MEC or keratinized epidermalcells are used as the cells of the present invention. The feeder layermethod may be used according to the known methods described, forexample, in Ueda M, The potential of Oral mucosal cells for culturedepithelium: a preliminary report. Ann. Plast. Surg., 35 (5), p498-504(1995), Rheinwald J G, Serial cultivation of strains of human epidermalkerationocytes: the formation of keratinizing colonies from singlecells., Cell., 6 (3), p 331-343 (1975).

Specific examples of the genus Escherichia available include Escherichiacoli K12/DH1 [Proc. Natl. Acad. Sci. USA, Vol. 60,160 (1968)]; JM103[Nucleic Acids Research, Vol 9, 309 (1981)]; JA221 [Journal of MolecularBiology, Vol. 12, p 517 (1978)]; HB101 [Journal of Molecular Biology,Vol. 41, p 459 (1969)]; C600 [Genetics, Vo. 39, 440 (1954)], DH5α[Inoue, H., Nojima, H. and Okayama, H., Gene, 96, p 23-28 (1990)], andDH10B [Proc. Natl. Acad. Sci. USA, Vol. 87, p 4645-4649 (1990)].Examples of the genus Bacillus available include Bacillus subtilis MI114[Gene, Vol. 24, 255 (1983); Journal of Biochemistry, Vol. 95,87(1984)].Examples of the genus Bifidus include Bifidobacterium longum,Bifidobacterium bifidum, and Bifidobacterium breve. Examples of lacticacid bacteria include the genus Lactobacillus, Streptoccoccus,Leuconostoc and Pediococcus.

Examples of yeast available include Saccharomyces cerevisiae AH22,AH22R⁻, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913 andNCYC2036, and Pichia pastoris.

When the virus is AcNVP, specific examples of insect cells availableinclude spodoptera frugiperda cell line (Sf cell) derived from larva,MG1 cells derived from midgut of Trichoplusia ni, High Five™ cellsderived from eovum of Trichoplusia ni, cells derived fromMamestrabrassicae and cells derived from Estigmena acrea. When the virusis BmNPV, cells derived from Bombyx mori N (BmN cell) are used. Examplesof the Sf cells available include Sf9 cell (ATCC CRL1711), Sf21 cell(Vaughn, J. L. et al., [In Vivo, 13, 213-217, (1977)].

DNA may be introduced according to the method described in SaibouKohgaku (Cell Technology), Supplement Edition 8, Sin Saibou KougakuJikken Purotokohru (Protocol of New Experimental Cell Technology),263-267 (1995), published by Shujun-sha Co, and Virology, Vo. 52, p456(1973).

DNA can be introduced into the genus Escherichia, for example, by themethod according to Proc. Natl. Acad. Sci. USA, Vol. 69, 2110 (1972) andGene, Vol. 17, 107 (1982). The genus Bacillus may be transformed, forexample, by the method according to Molecular & General Genetics, Vo.168, 111 (1979).

DNA can be introduced into yeast, for example, by the methods describedin Methods in Enzymology, Vol. 194, p 182-187 (1991) and Proc. Natl.Acad. Sci. USA, Vol. 75, p 1929 (1978).

DNA may be introduced into insect cells by the method, for example,described in Bio/Technology, Vol. 6, p 47-55 (1988).

Transformants into which an expression vector containing an NK4-encodingDNA is introduced can be thus obtained.

The preparation of the present invention comprises a cell containing anNK4-encoding DNA as well as a fibrous protein. The fibrous proteinserves as a carrier of the cells containing the NK4-encoding DNA in thecell-containing preparation of the invention. Containing the fibrousprotein permits the preparation of the invention to be formed intovarious shapes such as a sheet, sphere and tube, and the preparation maybe administered to all sites in the body. Examples of the fibrousprotein include collagen, keratin, fibroin, elastin and fibrin.Collagen, especially collagen type I as a major constituting element ofthe dermis and cartilage tissues of mammals is preferable in the presentinvention. The fibrous protein is safe to the living body since it is aprotein derived from organisms such as mammals.

It is preferable for the fibrous protein used in the present inventionto contain components of a culture medium suitable for culturing thecells of the invention. By including culture medium components in thefibrous protein, nutrients can be supplied to the cells in thepreparation of the present invention, leading to stable and longersurvival of the cells when the preparation of the present invention isadministered to the living body. As a result, the NK4 supply to thecancer tissue can be stabilized and extended for a longer period oftime.

A collagen gel containing the culture medium component can be preparedby the steps comprising: mixing the culture medium component suitablefor culturing the cells used in the present invention with the fibrousprotein, for example collagen type I; allowing the mixture to stand at30° C. to 37° C. for 15 minutes to 1 hour; and solidifying the mixtureinto a desired shape, for example, a sheet, sphere or tube. When thecollagen gel containing the culture medium component is formed into athick shape, for example, into a sheet, the collagen gel containing theculture medium component may be fixed on a mesh sheet comprising abiodegradable resin to prepare a collagen gel composite material inorder to maintain its strength and to simplify its handling. Althoughthe method for preparing the collagen gel composite material is notparticularly restricted, the collagen gel may be prepared on the meshsheet by pouring a mixture of collagen and culture medium component ontothe mesh sheet comprising the biodegradable resin.

A mesh sheet of a surgical suture made of a biodegradable polymermaterial is preferable for the mesh sheet of the biodegradable resin.Particularly, polyglycolic acid is preferable as the biodegradablepolymer in the present invention, and an example of the mesh sheet ofthe biodegradable resin is knitted-type VICRYL™ mesh; Ethicon, Inc., NewJersey.

While the cell-containing preparation of the present invention maycontain other additives such as delayed-release agents, isotonizingagents and pH control agents that are substantially admitted to add tothe conventional pharmaceutical preparations under the PharmaceuticalAffairs Law, it is preferable that the kind and content of them do notinterfere with proliferation, survival and/or NK4 secretion of the cellscontained in the preparation of the present invention, and the contentthereof is preferably within a range not impairing the object of theinvention.

These other additives may be incorporated into the culture medium inadvance when the collagen gel is prepared. Proteins such as gelatin,porous ceramics, polyamino acids, polylactic acid, chitin or chitinderivatives, and water-swellable polymer gel may be used for thedelayed-release agent. The isotonizing agent available includespolyethylene glycol and sodium chloride, and the pH control agentincludes phosphate salts and amino acids.

The collagen material containing the culture medium components may befurther compacted so as to have a high density, if necessary, forenhancing the strength thereof. Although there is no particularlimitation to the compacting method, a pressure is applied from abovethe collagen gel which is solidified using a medical tool such as arubber spatula or rubber cup, for example, silicone rubber cup.

While the preparation of the present invention is characterized bycomprising the fibrous protein containing the culture medium componentsas prepared above, for example, the collagen gel containing the culturemedium components, and the cells containing an NK4-encoding DNA, thecollagen gel containing the culture medium components may be eitherintegrated or not integrated with the cells containing the NK4-encodingDNA. The cells are dispersed in the collagen gel in the integratedpreparation, and such cell-dispersing collagen gel may be prepared bymixing the cells in the culture medium components in advance when thecollagen gel is prepared. The non-integrated preparation is defined tobe other than the integrated preparation, for example, the cells arelaminated on the surface of the collagen gel. Such cell-laminatedcollagen gel may be prepared by applying or seeding the cells on thesurface of the collagen gel followed by culturing the cell after thecollagen gel is prepared. Preferably, the cells and the collagen gel arenot integrated with each other, or the cells are particularly preferablylaminated on the surface of the collagen gel. The cell layer may be amonolayer or multilayer. Such layered structure permits thecell-containing layer to directly contact the administration site sothat NK4 secreted from the cells can be efficiently supplied to thecancer cells (see FIG. 1).

The NK4-encoding DNA may be introduced into the cells used in thepresent invention before being integrated or not integrated with thecollagen gel. Otherwise, the cells may be converted into the cellscontaining the NK4-encoding DNA through introduction of the NK4-encodingDNA after the cells are integrated or not integrated with the collagengel.

When the cells are integrated with the collagen gel in the presentinvention, a DNA is preferably introduced into the cell in advanceconsidering introduction efficiency of DNA into the host cell. On theother hand, when the cells are not integrated with the gel as apreferable embodiment of the present invention, i.e. when the cells arelaminated on the surface of the collagen gel, it is preferable tointroduce the NK4-encoding DNA into the laminated host cells, which arecultured on the collagen gel, using a vector such as a virus vector.

Although the culture medium contained in the fibrous protein material isnot particularly limited so long as it is a culture medium usually usedfor cultivation of animal cells when the host cell is an animal cell,examples of such medium available include an MEM medium [Science, Vol.,501 (1952)] containing about 5 to 20% of fetal bovine serum (FBS), DMEMmedium [Virology, Vol. 8, 396(1959)], RPMI 1640 medium [The Journal ofthe American Medical Association, Vol. 199, 519 (1967)], and 199 medium[Proceeding of the Society for the Biological Medicine, Vol. 73, 1(1950)]. The pH for cultivating the cell is preferably about 6 to 8. Thecells are usually cultivated at 30 to 40° C. for about 15 to 60 hours,and are aerated and stirred, if necessary. The culture conditions suchas the temperature, oxygen concentration and carbon dioxideconcentration may be appropriately determined depending on the cellsused.

While the culture medium used for cultivation is not particularlyrestricted so long as it contains carbon sources, nitrogen sources andinorganic salts necessary for the growth when the host is the genusEscherichia or the genus Bacillus, preferable examples thereof includesLB medium (manufactured by Nissui Pharmaceutical Co.), and M9 mediumcontaining glucose and casamino acid (Miller, Journal of Experiments inMolecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York,1972). If necessary, chemicals such as 3β-indolylacrylic acid may beadded in order to permit the promoter of the vector containing theNK4-encoding DNA to efficiently work. When the host is the genusEscherichia, the cells are usually cultured at 15 to 43° C. for about 3to 24 hours with optional stirring and aeration. When the host is thegenus Bacillus, the cells are usually cultured at 30 to 40° C. for about6 to 24 hours with optional stirring and aeration. When the host isyeast, examples of the culture medium include Baekholder minimum medium[Bostian, K. L. et al., Proc. Natl. Acad. Sci. USA, Vol. 77, p 4505(1980)], and SD medium containing 0.5% casamino acid [Bitter, G. A. etal., Proc. Natl. Acad. Sci. USA, Vol. 81, p 5330 (1984)]. The pH of theculture medium is preferably adjusted to about 5 to 8. Cultivation isusually continued for about 24 to 72 hours at about 20 to 35° C. withaeration and stirring, if necessary.

When the host is an insect cell, the culture medium used is Grace'sInsect Medium [T.C.C., Nature, Vol. 195, 788 (1962)] in which anadditive such as 10% of inactivated bovine serum is appropriately added.The pH of the medium is preferably controlled to about 6.2 to 6.4. Thecells are cultured at about 27° C. for about 3 to 5 days with optionalaeration and stirring.

The cell-containing preparation of the present invention ischaracterized in that NK4 is produced through incorporation ofNK4-encoding DNA or RNA into the host cell. NK4 produced by thecell-containing preparation of the present invention is a peptideencoded by (a) a DNA comprising a base sequence represented by SEQ IDNO:1 or 2, or encoded by (b) a DNA hybridizable with a DNA comprisingthe base sequence represented by SEQ ID NO:1 or 2 under stringentconditions, and exhibits an antagonist activity against HGF and aninhibitory action against angiogenesis. Examples of the particularlypreferable protein include (c) a peptide comprising an amino acidsequence represented by SEQ ID NO: 3 or 4, or (d) a peptidesubstantially identical to the peptide comprising the amino acidsequence represented by SEQ ID NO: 3 or 4 and having an amino acidsequence in which one to several amino acids are deleted, added orsubstituted, wherein the peptide has an antagonist activity against HGFand an inhibitory action against angiogenesis.

The basic operation of the gene engineering and bioengineering in thepresent invention are described in commercially available experimentaltextbooks by Idenshi Manyuaru (Gene Manipulation Manual), Kodansha Ltd.;Yasutaka Takagi, ed., Idenshi Jikken Sohsa Hou (Method of GeneManipultion), Kodansha Ltd.; Molecular Cloning Vol. 3, Methods inEnzymol.), 194 (1991); and Jikkenn Igaku (Experimental Medicine),supplement edition, Method of Gene Experiment using Yeast, Yohdo-sha Co.(1994).

The cell-containing preparation of the present invention can be formedinto various shapes such as a sheet, sphere or tube suitable foradministering to various sites by adding a fibrous protein such ascollagen as a carrier, and is able to administer by embedding intosubcutaneous and intramuscular sites. For example, the cancer tissueitself may be covered with the sheet by forming the preparation into asheet to enable NK4 to supply to respective cells in the entire cancertissue in proper quantities. Metastasis and recurrence of the cancerafter the surgery may be prevented by applying the sheet so as to coverthe site where the cancer tissue has been removed through a surgicaloperation.

Although the dosage of the cell-containing preparation of the presentinvention may be controlled depending on the kind of diseases to becured, condition, age and body weight of patients, and administrationsites, it is usually 0.01 to 2000 mg, preferably 0.1 to 100 mg, as theweight of the NK4-encoding DNA for a patient with a body weight of 60kg. The preparation may be administered to other animals by convertingthe dosage per 60 kg of the body weight.

The cell-containing preparation of the present invention can be used aspreventive and therapeutic agents of diseases caused by cancers and/orangiogenesis, and as inhibitors for cancer metastasis. Accordingly, thecell-containing preparation of the present invention may be used forinhibition of invasion, growth and metastasis of tumors, induction ofapoptosis, and/or inhibition of angiogenesis. The present invention alsoprovides a method for inhibiting invasion, growth and metastasis oftumors, induction apoptosis and/or inhibition of angiogenesis using thecell-containing preparation of the present invention.

Examples of the cancer as the target disease of the present inventioninclude lung cancer, ovarian cancer, pancreatic cancer, stomach cancer,gall bladder cancer, kidney cancer, prostate cancer, breast cancer,esophageal cancer, liver cancer, oral cavity cancer, colon cancer, largeintestine cancer, uterine cancer, bile duct cancer, islet cell cancer,adrenal cortex cancer, bladder cancer, testicular cancer, testiculartumor, thyroid cancer, skin cancer, malignant carcinoid tumor, malignantmelanoma, osteosarcoma, soft tissue sarcoma, neuroblastoma, Wilms'tumor, retinoblastoma, melanoma and glioma. Among them, ovarian cancer,pancreatic cancer, stomach cancer, gall bladder cancer, kidney cancer,prostate cancer, breast cancer, esophageal cancer, liver cancer, oralcavity cancer, colon cancer, large intestine cancer, sarcoma, melanomaand glioma are preferable, and ovarian cancer, pancreatic cancer,stomach cancer and gall bladder cancer are particularly preferable.

Examples of diseases due to angiogenesis include rheumatoid arthritis,psoriasis, Osler-Webber syndrome, myocardial vasculogenesis,telangiectasia, hemophilic arthritis, ocular vasculogenesis (forexample, diabetic retinopathy, retinopathy of prematurity, age-relatedmacular degeneration, corneal transplant rejection, neovascularglaucoma, retrolental fibroplasia, or perosis), vascular fibroma, benigntumor (for example, hemangioma, acoustic neuroma, neurof ibroma,trachoma, pyogenic granuloma), tumor of hemopoietic organ includingleukemia, solid tumor, tumor metastasis, granulation wound, and thelike. The cell-containing preparation of the present invention may beused in combination with surgical treatment, radiotherapy andchemotherapy. Excision of tumors is one of the surgical treatment.Radiotherapy include γ-ray, X-ray, microwaves or electron beanirradiation. Chemotherapy includes administration of antitumor agents.Examples of the antitumor agent include alkylation agents, variousmetabolic antagonists, antitumor antibiotics, antitumor plant extractsand BRM (biological response modulators).

The effect of the cell-containing preparation of the present inventionas anticancer agents and inhibitors of cancer metastasis, i.e.antagonist activity against HGF and inhibitory action againstangiogenesis involved in the preparation of the present invention may bedetermined according to the known methods, for example, according to thedetermination method to be described hereinafter.

EXAMPLES

While examples of the invention are described hereinafter, thedisclosure below represents only preferable examples, and does notrestrict the technical scope of the present invention in any sense.

Production Example Preparation of Cell-Containing Preparation

(1) Preparation of NK4 cDNA

mRNA was isolated from subcutaneous tissue cells of Wister rat or OMECusing ISOGEN-LS (NipponGene Co., Ltd., Toyama, Japan), and the mRNA wasused for RT-PCR(reverse transcription/polymerase chain reaction) toisolate NK4 cDNA. Specifically, 0.5 μl of mRNA solution (150 ng ofmRNA), and 5 μl of 10×RT-PCR solution (500 mM KC1, 100 mM Tris-HCl (pH9.0), 1% Triton X-100, 15 mM MgCl₂), 4 μl of dNTP (2.5 mM), 2 μl ofprimerl (10 mM), 2μl of primer 2 (10 mM), 0.5 μl of Taq polymerase(Takara), 0.5 μl of RNasin (Promega), 0.5 μl of reverse transcriptase(Takara) and 35.2 μl of DEPC-treated H₂O were mixed. The reversetranscription reaction was performed at 42° C. for 30 minutes and at 95°C. for 5 minuets, and a cycle of 94° C. for 30 seconds, 55° C. for 1minute and 72° C. for 1 minute was repeated 40 times, followed by areaction at 72° C. for 7 minutes to obtain NK4 cDNA. NK4 cDNA thusobtained was cloned into pCRII™ vector using TA Cloning Kit (Invitrogen)to obtain pCRII/NK4. The primer used was the DNA fragment represented bySEQ ID NO:5 or 6.

(2) Construction of Recombinant Expression Vector

NK4 cDNA integrated into pCRII vector prepared in the above (1) was cutwith a restriction enzyme KpnI/SpeI, and the cut-end was blunted bytreating with T4 DNA polymerase (Takara). The NK4 cDNA fragment obtainedwas treated with a restriction enzyme XhoI, mixed with human adenovirusvector V (E1a deficient, E3 partially deficient) having a cut-end thathas been blunted, and an NK4 expression vector Ad-NK4 was obtained byligating the fragment with the vector using T4 DNA ligase (FIG. 2).

(3) Establishment of OMEC

Intraoral tissues were sampled from Wister rat with age 3 to 6 weeks andwere subdivided. The tissue pieces were immersed twice in PBS (pH7.4.4,Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing antibiotics(1000 U/ml of penicillin G potassium, 1 mg/ml of kanamycine and 2.5μg/ml of amphotericin B). The tissue after immersion was immersed againin a DMEM culture medium (Gibco Laboratories Inc., Grand Island, N.Y.)containing 0.2% dispase (Sigma-Aldrich Co., St. Louis, Mo.).Subsequently, the tissue was treated at room temperature for 30 minutesusing a solution containing 0.25% of trypsin and 5 mM EDTA, followed bywashing with a DMEM culture medium containing 10% of FBS (CSL Ltd.,Victoria, Australia). The sample tissue obtained was stirred for 30minutes in the DMEM culture medium containing 5% of FBS to release thecells, and the free OMEC cells were obtained by filtering with a filterwith a pore size of 50 μm.

After treating Swiss 3T3 cells (Dainippon Pharmaceutical Co., Ltd.;Osaka, Japan) with 4 μg/ml of mitomycin C (Wako Pure ChemicalIndustries, Tokyo, Japan) for 2 hours, 1×10⁵ cells were seeded on eachwell (Costar Inc., NY) of 6-well plate filled with EFM culture medium(DMEM culture medium: Ham's F culture medium (Nihonseiyaku, Tokyo,Japan)=3:1) containing 10% CO₂. The 10% CO₂-containing EFM culturemedium was supplemented with 5% FBS, 5 μg/ml insulin (Wako Pure ChemicalIndustries), 5 μg/ml transferrin (Wako Pure Chemical Industries), 2×10⁻⁹M triiodotyrosine (Sigma-Aldrich Co.), 10 ng/ml cholera toxin(Sigma-Aldrich Co.), 0.5 μg/ml hydrocortisone (Wako Pure ChemicalIndustries), 100 U/ml penicillin, 0.1 mg/ml kanamycine and 0.25 mg/ml ofamphotericin B.

Subsequently, 1×10⁵ of free OMEC was seeded on each well. On day threeafter seeding the cell, 10 ng/ml of epidermal growth factor (humanrecombinant epidermal growth factor: Wako Pure Chemical Industries) wasadded to each well. After confirming that OMEC had grown to confluenceafter 7 to 10 days, the cells were subjected to passage culture. Thesecond or third passage culture cells were harvested as establishedcells.

(4) Preparation of Collagen Gel

Knitted mesh sheet VICRYL™ (polyglactin 910; Nithicon, Inc., New Jersey)was placed on each well of a 24-well plate (Greiner Bio-one Co., Ltd.,Frickenhausen, Germany), and a mixed solution of 0.5 ml of 10%FBS-containing DEM culture medium and 0.5 ml of collagen type I solution(0.3% by mass; CELLGEN, Koken Corp., Tokyo, Japan) suspended in the sameculture medium was poured onto the mesh sheet. A collagen-VICRYL™ meshcomposite material was prepared by allowing the mesh sheet to stand at37° C. for 30 minutes. The collagen gel on the composite material wascompacted by compression using a silicone cup.

(5) Preparation of Collagen Gel-Adhered OMEC Sheet

Established OMEC obtained in the above (3) was treated to free cellsusing 0.05% trypsin-EDTA. The free cells obtained were seeded on thesurface of the collagen gel obtained in the above (4) in a density of2×10⁵ cells/cm². An OMEC layer was formed on the surface of the collagengel by cultivation at 37° C. for 2 to 3 days. FIG. 3 shows a crosssection of the collagen gel-adhered OMEC sheet photographed by SEM(scanning electron microscope). It was confirmed from FIG. 3 that thecollagen gel-adhered OMEC sheet comprises three layers of an OMEC celllayer, a collagen gel layer and a VICRYL™ mesh sheet. An electronmicroscope JSM-840A manufactured by JOEL Co. was used for the SEManalysis.

(6) Introduction of Ad-NK4 into OMEC

Five hundreds μl of 2% FBS-containing DMEM culture medium was added toeach well of 24-well plate in which collagen-adhered OMEC obtained inthe above (5) was contained, and OMEC was transfected with Ad-NK4prepared in the above (2) at 37° C. for 2 hours and at 10 to 200 MOI(multiplicity of infection) per 500 μl of the culture medium. Theculture supernatant was removed after transfection to obtain acell-containing preparation (Production Example 1). Collagen gel-adheredOMEC was also transfected with E1a and E1b expressing lacZ gene ofEscherichia coli, and with E3 deficient adenovirus vector (Ad-lacZ: H.Ueno, University of Occupational and Environmental Health, Fukuoka,Japan) to prepare a sample of Comparative Example 1.

Test Example 1 Confirmation of Production Ability of NK4 of OMEC intoWhich DNA Encoding NK4 has been Introduced

OMEC (2×10⁵ cells) was seeded in each well of a 12-well plate (GreinerBio-one Co., Ltd.) coated with collagen type I. After adding 1 ml of 2%FBS-containing DMEM culture medium, the cells were cultivated for 72hours, followed by transfecting with Ad-NK4 at 10, 50 and 100 MOIrelative to 500 μl of the culture medium (OMEC into which Ad-NK4 isintroduced). The supernatant of the culture medium was removed after theinfection, and 1 ml of DMEM containing 2% FBS was added to each well.The culture supernatant was taken out at every 48 hours after theinfection to measure the amount of secreted NK4 in the culturesupernatant. OMEC not infected with Ad-NK4 was also cultured as acontrol, and the amount of secreted NK4 was measured. The concentrationof NK4 was measured using IMMUNUS human HGF enzyme immunoassay kit(Institute of Immunology, Tokyo, Japan).

The results are shown in FIG. 4. The amount of secretion of NK4 was themaximum at MOI 100 and 200 in Ad-NK4-introduced OMEC, and the amount wasgradually decreased independent of MOI. No secretion of NK4 was observedin OMEC into which Ad-NK4 was not introduced. Each measured value wasexpressed by “average value±SD”.

Test Example 2 Confirmation of Proliferation Ability of OMEC into WhichDNA Encoding NK4 was Introduced

OMEC (5×10⁴ cells) were seeded on each well of 24-well plate coated withcollagen type I. After culturing for 72 hours, OMEC was transfected withAd-NK4 at 100 MOI (OMEC in which Ad-NK4 was introduced). The culturesupernatant was removed after transfection, and 500 μl of the FEMculture medium was added to each well. After 24 or 48 hours, the cellswere separated using 0.05% EDTA, and the number of the cells was countedwith a cell counter (Coulter counter: Beckman Coulter Inc., CA). OMECnot transfected with Ad-NK4 (OMEC into which no Ad-NK4 was introduced)was also cultivated as a control, and the cell number was counted. Eachmeasured value was expressed by “mean value±SD”.

The results are shown in FIG. 5. No difference was observed between theproliferation ability of OMEC in which Ad-NK4 was introduced and that ofOMEC in which Ad-NK4 was not introduced on the collagen gel. From thecombined results of this Test Example land Test Example 2, it wasconfirmed that proliferation ability on the collagen gel was notaffected even through transformation of OMEC with the virus vector andsecretion of NK4.

Test Example 3 Confirmation of Inhibitory Effect of Cancer Invasion byNK4 Produced from OMEC into Which DNA Encoding NK4 is Introduced

OMEC (1×10⁵ cells) were seeded on 12-well plate coated with collagentype I. After 72 hours of cultivation, OMEC was transfected with Ad-NK4100 MOI (OMEC into which Ad-NK4 was introduced). The culture supernatantwas removed after the infection, and 1 ml of DMEM containing 2% FBS wasadded to each well. The culture supernatant was sampled 3 or 4 daysafter transfection, and this supernatant (NK4-sup) was used for theinvasion inhibition test. Used for the invasion inhibition test was24-well Matrigel Invasion double chamber (Becton Dikinson, Bedfold,Mass.).

HGF-producing human fibroblast cells (MRC-5: RIKEN Cell Bank, Ibaraki,Japan) were seeded in 10% FBS-containing DMEM contained in the outer cupof Matrigel invasion double chamber at a density of 1.5×10⁵ cells/cm².After 24 hours of cultivation, the culture medium was replaced with 2%FBS-containing DMEM. DMEM containing 2% FBS (control) and spleen cancercells (SUIT-2 or AsPC-1: H. Iguchi, National Kyushu Cancer Center,Fukuoka, Japan) suspended in the above NK4-sup were seeded at a densityof 5×10⁴/cup in the inner cups of Matrigel invasion double chamber,respectively. After 24 hours of cultivation, the cells were stained withhematoxin-eosin to count the number of the invaded spleen cancer cells.Five fields of vision were randomly selected under the microscope tomeasure the number of the invaded cells, and the number was expressed interms of “average value±SD”.

The results are shown in FIG. 6. While almost no invasion was observedin the absence of MRC-5 (MRC-5(−)) in both the spleen cancer cellssuspended in 2% FBS-containing DMEM (control) and the spleen cancercells suspended in NK4-sup irrespective of the kind of the spleen cells,invasion of the spleen cells was observed in the presence of MRC-5(MRC-5(+)) as compared with the result obtained in the absence of MRC-5(MRC-5(−)), and the number of invaded cells was remarkably increased inSUIT-2 cells. In contrast, the number of the invaded spleen cancer cellswas remarkably inhibited for the SUIT-2 and AsPC-1 cells when the spleencancer cells were suspended in the NK4-sup.

Example 1 Production of NK4 for Cell-Containing Preparation

AsPC-1 cells (3×10⁶ cells) suspended in 50 μl of DMEM were transplantedinto the muscle at the left flank of a 4-weeks old nude mouse (BALB/cnu/nu, Kyudo Co., Ltd., Saga, Japan) to form a tumor. Fourteen daysafter implantation of AsPC-1 cells, the cell-containing preparationsprepared in Production Example 1 (Production Example 1 group: n=3) andComparative Example 1 (Comparative Example 1 group: n=3) weresubcutaneously transplanted respectively at the tumor-forming sites asshown in FIG. 1. A group was also prepared by transplanting only AsPC-1cells as a control (control group: n=3). Tumors of each nude mouse inProduction Example 1 group and Comparative Example 1 group were sampledtogether with surrounding tissues on day 4 after implantation. The tumorsampled from each group was washed once with PBS and homogenized usingRIPA buffer [1% RIPA (UPSTATE, NY), 1 mM sodium orthovanadate (Wako PureChemical Industries), 1 mM phenylmethylsulfonyl fluoride (Wako PureChemical Inductries)] to obtain an extraction solution of each tumor.The amount of NK4 in this tumor extraction solution was measured byELISA method. The serum was also sampled from each mouse in ProductionExample 1 group, and the amount of NK4 in the serum was measured by thesame method. Each measured value was expressed in terms of “averagevalue±SD”.

The results are shown in FIG. 7. While the amount of NK4 from thecontrol group and Comparative Example 1 group was not more than 0.3ng/total protein (g), the amount of NK4 in the tumor extraction solutionfrom the Production Example 1 group was 155.85±60.83 ng/total protein(g). NK4 was not detected in the serum of Production Example 1 group. Itwas confirmed from these results that NK4 secreted from thecell-containing preparation of Production Example 1 was supplied to thetumor of the mouse implanted with the preparation in Production Example1, and that the NK4 was locally and efficiently supplied to the targettumor.

Example 2 Inhibition of Growth of Cancer using Cell-ContainingPreparation

Tumors were formed in nude mice by the same method as in Example 1.Three days after implantation of AsPC-1 cells, the cell-containingpreparation prepared in Production Example 1 (Production Example 1group: n=5) or Comparative Example 1 (Comparative Example 1 group: n=5)was subcutaneously transplanted at the tumor-forming site as shown inFIG. 1. A group (control group: n=5) in which only AsPC-1 cells weretransplanted was also prepared as a control. On day 3 and 5 afterimplantation of the preparation in Production Example 1 or ComparativeExample 1, the volume of the tumor in nude mouse was measured using agauge (caliper). The volume of the tumor was defined by the followingequation. Each measured value was expressed in terms of average value±SD(p<0.05).

Volume of tumor (mm2)=0.52×(width (mm))²×length (mm)

The results are shown in FIG. 8. The tumor volume was remarkablyincreased with the lapse of time in Comparative Example 1 group andcontrol group. On the contrary, the increase of the tumor volume wasinhibited in Production Example 1 group. These results clearly show thatthe cell-containing preparation according to Production Example 1 has aninhibitory effect of tumor proliferation.

Example 3 Inhibition of Angiogenesis using Cell-Containing Preparation

Tumors were formed in nude mice by the same method as in Example 1.Three days after implantation of AsPC-1 cells, the cell-containingpreparation prepared in Production Example 1 (Production Example 1group: n=4) and in Comparative Example 1 (Comparative Example 1 group:n=4) were subcutaneously transplanted at the tumor-forming site as shownin FIG. 1. A group (control group: n=3) to which only AsPC-1 cells wereimplanted was also prepared as a control. On day 14 after implantationof AsPC-1 cells, the tumor sampled from each group was subjected to animmnohistochemical staining, and the number of newborn blood vessels wasmeasured. The immnohistochemical staining will be described below. Atissue section sampled from each group was fixed with paraffin, and arabbit polyclonal antibody against von Willebrand factor (DAKO Co., CA)was adhered to each tissue section, which was stained withDAB-peroxidase complex. The number of newborn blood vessels was countedunder a microscope (×200). Each measured value was expressed in terms ofaverage value±SD (p<0.05).

The results are shown in FIG. 9. Remarkable angiogenesis was observed inComparative Example 1 group and control group. On the contrary, increaseof the number of newborn blood vessels was inhibited in ProductionExample 1 group. These results show that the preparation according toProduction Example 1 has a angiogenesis inhibitory effect.

INDUSTRIAL APPLICABILITY

The cell-containing preparation of the present invention is an effectivetherapeutic agent for inhibiting the metastasis of primary tumors orcancers, and is safe to the body. In particular, NK4 having actions forinhibiting the growth or metastasis of cancers can be more efficientlysupplied to cancer cells according to the present invention. The presentinvention provides a method for inhibiting growth of primary tumors, amethod for inhibiting metastasis of cancers, or a method for inhibitingangiogenesis using the cell-containing preparation of the presentinvention.

1. A cell-containing preparation comprising a cell which has a DNAhaving a base sequence represented by SEQ ID NO: 1 or 2 or a DNAhybridizable with a DNA having a base sequence represented by SEQ ID NO:1 or 2 under stringent conditions and a fibrous protein.
 2. Thecell-containing preparation according to claim 1, wherein the cell is anepithelial cell of the oral mucosa, a skin cell or a fibroblast.
 3. Thecell-containing preparation according to claim 1, wherein the fibrousprotein is collagen.
 4. The cell-containing preparation according toclaim 1, wherein the cells are deposited on the surface of the fibrousprotein.
 5. The cell-containing preparation according to claim 1,wherein the cell is a transformant.
 6. The cell-containing preparationaccording to claim 5, wherein the transformant is transformed with arecombinant expression vector.
 7. The cell-containing preparationaccording to claim 6, wherein the recombinant expression vector isadeno-associated virus (AAV), retrovirus, poxvirus, herpes virus, herpessimplex virus, lentivirus (HIV), Sendai virus, Epstein-Barr virus (EBV),vaccinia virus, polio virus, sindbis virus, SV40 or plasmid.
 8. Thecell-containing preparation according claim 1 capable of forming apeptide encoded by a DNA having a base sequence represented by SEQ IDNO:1 or 2, or by a DNA hybridizable with a DNA having a base sequencerepresented by SEQ ID NO: 1 or 2 under stringent conditions.
 9. Thecell-containing preparation according to claim 1 further containing amesh sheet comprising a biodegradable resin.
 10. The cell-containingpreparation according to claim 9, wherein the biodegradable resin ispolyglycolic acid.
 11. The cell-containing preparation according toclaim 1, which is an anticancer agent or a cancer metastasis inhibitor.12. The cell-containing preparation according to claim 11, which is ananticancer agent or a metastasis inhibitor for ovarian cancer,pancreatic cancer, stomach cancer, gall bladder cancer, kidney cancer,prostate cancer, breast cancer, esophageal cancer, liver cancer, oralcavity cancer, colon cancer, large intestine cancer, sarcoma, glioma ormelanoma.
 13. The cell-containing preparation according to claim 1,which is an angiogenesis inhibitor.
 14. A method for inhibiting growth,invasion and metastasis of cancers or for inhibiting angiogenesis, whichcomprises administering the cell-containing preparation according toclaim 1 to a mammal.
 15. A method for producing a cell-containingpreparation, which comprises culturing a cell on the surface of afibrous protein and transforming the cultured cells with a recombinantexpression vector comprising a DNA having a base sequence represented bySEQ ID NO:1 or 2, or with a recombinant expression vector comprising aDNA hybridizable with a DNA having a base sequence represented by SEQ IDNO:1 or 2 under stringent conditions.
 16. A method for producing acell-containing preparation, which comprises preparing a fibrous proteinsheet by coating a fibrous protein onto a mesh sheet comprising abiodegradable resin; culturing a cell on the surface of the fibrousprotein sheet obtained; and transforming the cultured cells with arecombinant expression vector comprising a DNA having a base sequencerepresented by SEQ ID NO:1 or 2, or with a recombinant expression vectorcomprising a DNA hybridizable with a DNA having a base sequencerepresented by SEQ ID NO:1 or 2 under stringent conditions.
 17. A methodfor producing a cell-containing preparation, which comprisestransforming the cells with a recombinant expression vector comprising aDNA having a base sequence represented by SEQ ID NO:1 or 2, or with arecombinant expression vector comprising a DNA hybridizable with a DNAhaving a base sequence represented by SEQ ID NO:1 or 2 under stringentconditions, and mixing the resulting transformant cells with a fibrousprotein.